Members of the protein tyrosine phosphatase (PTP) super family contain the highly conserved active site motif, Cys-x5-Arg (Cx5R), and are key mediators of a variety of cellular processes including growth, differentiation, motility, metabolism, and programmed cell death. This proposal focuses on the mitochondrial phosphatase, PTPMT1 (formerly PLIP), the first resident mitochondrial phosphatase to be identified and functionally linked to energy metabolism in the cell. PTPMT1 is a highly conserved phosphatase with over 60 orthologs throughout the Animalia, Plantae, Protista, and Eubacteria kingdoms. There is no precedent for PTPs in the mitochondrion, and no known mitochondrial signaling pathways involving reversible phosphorylation. We, therefore, performed several analyses of cells in which PTPMT1 had been ablated. A lipidomic analysis comparing the lipid profile of PTPMTI-ablated versus wild type cells pointed to phosphatidylglycerol phosphate (PGP) as the in vivo substrate for PTPMT1. PGP is an intermediate in cardiolipin biosynthesis, a key component of inner mitochondrial membranes. We propose to: 1) Synthesize PGP for in vitro analyses to demonstrate that PTPMT1 is a PGP phosphatase. We will also use this substrate with a variety of other phosphatases to determine if the ability to convert PGP to PG is an enzymatic activity unique to PTPMT1. Conversely, the ability of PTPMT1 to utilize other lipid phosphate substrates will also be assessed. 2) Address whether PGP phosphatase activity is conserved in selected orthologues. 3) Address the function of PTPMT1 in the inner mitochondrial membrane. We propose to quantitate the effects of PTPMT1 expression on rates of cardiolipin biosynthesis, as well as examine the mitochondrial morphology and bioenergetic status of PTPMT1 deficient cells. 4) Determine the structure of PTPMT1 itself and in combination with PGP. These experiments will allow a detailed understanding of how PTPMT1 exhibits such unusual phospholipid substrate specificity. Our results will shed light on the possible roles of PTPMT1 in various diseases where reduced cardiolipin has been reported, such as ischemia, heart failure and diabetes, and provide insight into novel points of therapeutic intervention for these diseases.